Comparison of proton channel, phagocyte oxidase, and respiratory burst levels between human eosinophil and neutrophil granulocytes.

Robust production of reactive oxygen species (ROS) by phagocyte NADPH oxidase (phox) during the respiratory burst (RB) is a characteristic feature of eosinophil and neutrophil granulocytes. In these cells the voltage-gated proton channel (Hv1) is now considered as an ancillary subunit of the phox needed for intense ROS production. Multiple sources reported that the expression of phox subunits and RB is more intensive in eosinophils than in neutrophils. In most of these studies the eosinophils were not isolated from healthy individuals, and a comparative analysis of Hv1 expression had never been carried out. We performed a systematic comparison of the levels of essential phox subunits, Hv1 expression and ROS producing capacity between eosinophils and neutrophils of healthy individuals. The expression of phox components was similar, whereas the amount of Hv1 was ∼ 10-fold greater in eosinophils. Furthermore, Hv1 expression correlated with Nox2 expression only in eosinophils. Additionally, in confocal microscopy experiments co-accumulation of Hv1 and Nox2 at the cell periphery was observed in resting eosinophils but not in neutrophils. While phorbol-12-myristate-13-acetate-induced peak extracellular ROS release was ∼ 1.7-fold greater in eosinophils, oxygen consumption studies indicated that the maximal intensity of the RB is only ∼ 1.4-fold greater in eosinophils. Our data reinforce that eosinophils, unlike neutrophils, generate ROS predominantly extracellularly. In contrast to previous works we have found that the two granulocyte types display very similar phox subunit expression and RB capacity. The large difference in Hv1 expression suggests that its support to intense ROS production is more important at the cell surface.

Reduction of Bcr-Abl function leads to erythroid differentiation of K562 cells via downregulation of ERK.

The chimeric bcr-abl gene encodes a constitutively active tyrosine kinase that leads to abnormal transduction of growth and survival signals leading to chronic myeloid leukemia (CML). According to our previous observations, in vitro differentiation of several erythroid cell lines is accompanied by the downregulation of extracellular signal-regulated kinases (ERK)1/2 mitogen-activated protein kinase (MAPK) activities. In this work we investigated whether ERKs have a decisive role in either the erythroid differentiation process or apoptosis of bcr-abl+ K562 cells by means of direct (MEK1/2 inhibitor UO126) and indirect (reduced Bcr-Abl function) inhibition of their activities. We found that both Gleevec and UO126 induced hemoglobin expression. Gleevec treatment reduced the phosphorylation of Bcr-Abl, ERK and STAT-5 for up to 24 h, decreased Bcl-XL levels, and induced caspase-3-dependent apoptosis. In contrast, UO126 treatment resulted in only a transient decrease of ERK activity and did not induce cell death. For studying the effect of reduced Bcr-Abl function on erythroid differentiation at the level of the bcr-abl transcript, we applied the siRNA approach. Stable degradation of bcr-abl mRNA was achieved by using a retroviral vector with enhanced green fluorescent protein (EGFP) reporter. Despite a high (>90%) transduction efficiency we detected only a transient decrease in Bcr-Abl protein and in phosphorylated ERK1/2 levels. This transient change in Bcr-Abl signaling was sufficient to induce hemoglobin expression without significant cell death. These results suggest that by transiently reducing Bcr-Abl function it is possible to overcome the differentiation blockade without evoking apoptosis in CML cells and that reduced ERK activity may have a crucial role in this process.

Calcium signalling is altered in myeloid cells with a deficiency in NADPH oxidase activity.

The relation of O(2.-)-production and Ca2+ homeostasis was investigated in PLB-985 cell lines and neutrophilic granulocytes from peripheral blood. In differentiated wild-type PLB-985 cells, a high level of O(2.-)-production was associated with a significant decrease in the membrane potential and the inhibition of capacitative Ca2+ entry. These correlations were not observed in gp91phox -/- cells or in cells transfected with a non-functional mutant of gp91phox (Thr341Lys). Membrane depolarization and inhibition of Ca2+ entry reappeared in cells transfected with wild-type gp91phox. These experiments demonstrate that inhibition of Ca2+ entry depends on the presence of a functional NADPH oxidase. The Ca2+ signal induced by stimulation of chemotactic receptors also showed remarkable differences: [Ca2+]ic in the sustained phase was higher in gp91phox-/- than in wild-type cells. Alteration of the Ca2+ signal was reproduced by treating peripheral blood neutrophils with the NADPH oxidase inhibitor diphenylene-iodonium. It is concluded that the deficiency in O(2.-)-production is accompanied by significant alterations of Ca2+ homeostasis in myeloid cells.

Role of prenylation in the interaction of Rho-family small GTPases with GTPase activating proteins.

The role of prenylation in the interaction of Rho-family small GTPases with their GTPase activating proteins (GAPs) was investigated. Prenylated and nonprenylated small GTPases were expressed in Sf9 insect cells and Escherichia coli, respectively. Nucleotide binding to and hydrolysis by prenylated and nonprenylated proteins were identical, but three major differences were observed in their reactions with GAPs. (1) Membrane-associated GAPs accelerate GTP hydrolysis only on prenylated Rac1 and RhoA, but they are inactive on the nonprenylated form of these proteins. The difference is independent of the presence of detergents. In contrast to Rac1 and RhoA, nonprenylated Cdc42 is able to interact with membrane-localized GAPs. (2) Full-length p50RhoGAP and p190RhoGAP react less intensely with nonprenylated Rac1 than with the prenylated protein, whereas no difference was observed in the reaction of isolated GAP domains of either p50RhoGAP or Bcr with the different types of Rac1. (3) Fluoride exerts a significant inhibitory effect only on the interaction of prenylated Rac1 with the isolated GAP domains of p50RhoGAP or Bcr. The effect of fluoride is not influenced by addition or chelation of Al(3+). This is the first detailed study demonstrating that prenylation of the small GTPase is an important factor in determining its reaction with GAPs. It is suggested that both intramolecular interactions and membrane targeting of GAP proteins represent potential mechanisms regulating Rac signaling.

Characterization of membrane-localized and cytosolic Rac-GTPase-activating proteins in human neutrophil granulocytes: contribution to the regulation of NADPH oxidase.

We have investigated the intracellular localization and molecular identity of Rac-GTPase-activating proteins (Rac-GAPs) in human neutrophils. Immunoblot analysis detected the presence of both p190RhoGAP and Bcr mainly in the cytosol. An overlay assay performed with [gamma-(32)P]GTP-bound Rac revealed dominant GAP activity related to a 50 kDa protein both in the membrane and cytosol. This activity could be identified by Western blotting and immunoprecipitation with specific antibody directed against the GAP domain of p50RhoGAP. Using a semirecombinant or fully purified cell-free activation assay of the Rac-activated enzyme NADPH oxidase, we demonstrated the regulatory effect of both the membrane-localized and soluble GAPs. We suggest that in neutrophil granulocytes Rac-GAPs have redundant function and represent suitable targets for both the up-regulation and down-regulation of the NADPH oxidase.

Chronic granulomatous disease: more than the lack of superoxide?

Chronic granulomatous disease (CGD) is an inherited disease characterized by severe and recurrent bacterial and fungal infections manifested in most cases in early childhood. Phagocytic cells of CGD patients are unable to produce superoxide anions, and their efficiency in bacterial killing is significantly impaired. Recent work has shown alterations in the electrophysiological properties of CGD granulocytes, which might contribute to the pathogenesis of the disease. The new aspects that we discuss in this review concern the proton channel function of gp91phox (the electron-transporting subunit of the NADPH oxidase) and the electrogenic activity of the active enzyme complex, which can affect the transmembrane trafficking of several ions. Based on the reviewed data, we also propose a hypothesis that the absence of a functional NADPH oxidase in CGD neutrophils could result in altered ion compositions within intracellular and intraphagosomal spaces during the process of phagocytosis.

Identification of renox, an NAD(P)H oxidase in kidney.

Oxygen sensing is essential for homeostasis in all aerobic organisms, but its mechanism is poorly understood. Data suggest that a phagocytic-like NAD(P)H oxidase producing reactive oxygen species serves as a primary sensor for oxygen. We have characterized a source of superoxide anions in the kidney that we refer to as a renal NAD(P)H oxidase or Renox. Renox is homologous to gp91(phox) (91-kDa subunit of the phagocyte oxidase), the electron-transporting subunit of phagocytic NADPH oxidase, and contains all of the structural motifs considered essential for binding of heme, flavin, and nucleotide. In situ RNA hybridization revealed that renox is highly expressed at the site of erythropoietin production in the renal cortex, showing the greatest accumulation of renox mRNA in proximal convoluted tubule epithelial cells. NIH 3T3 fibroblasts overexpressing transfected Renox show increased production of superoxide and develop signs of cellular senescence. Our data suggest that Renox, as a renal source of reactive oxygen species, is a likely candidate for the oxygen sensor function regulating oxygen-dependent gene expression and may also have a role in the development of inflammatory processes in the kidney.

Role of different Ca2+ sources in the superoxide production of human neutrophil granulocytes.

The role of different Ca2+ sources in the activation of the NADPH oxidase was investigated in human neutrophil granulocytes. Selective depletion of the stimulus-responsive intracellular Ca2+ -pool and the consequent opening of the store-dependent Ca2+ channel of the plasma membrane was achieved with thapsigargin, an inhibitor of microsomal Ca2+ -ATPase. Low concentration (10-100 nM) of thapsigargin did not induce any O2*- -production, indicating that elevation of [Ca2+]ic to similar level and probably via similar route as following stimulation of chemotactic receptors, by itself is not sufficient to activate the NADPH oxidase. In significantly higher concentration (1-10 microM) thapsigargin did induce O2*- -generation but this effect was not the result of elevation of [Ca2+]ic. In the absence of external Ca2+ a gradual decrease of the responsive Ca2+ pool was accompanied by a gradual decrease of the rate and duration of the respiratory response stimulated by formyl-methionyl-leucyl-phenylalanin. Maximal extent of receptor-initiated O2*- -production could only be obtained when the intracellular [Ca2+] was higher than the resting level. Under this condition Ca2+ originating from intracellular or external source was equally effective in supporting the biological response.

[Medical importance of GTP-binding proteins]. / Kis GTP-kötö fehérjék orvosi jelentösége.

Small GTP-binding proteins consist of one single, 21 kD polypeptide chain. These regulatory proteins are active in the GTP-bound but inactive in the GDP-bound form. They are involved in the regulation of several important cell functions, such as cell division, cell motility, shape changes, intracellular membrane traffic, transport of macromolecules between cytoplasm and nucleus, assembly of enzyme complexes. Recently several mutations have been described which modify the function of small G-proteins resulting in typical pathological changes. These diseases and the molecular alterations involved in their pathomechanism are summarized in this review.

Differential effects of tyrosine kinase inhibitors and an inhibitor of the mitogen-activated protein kinase cascade on degranulation and superoxide production of human neutrophil granulocytes.

The effects of two different tyrosine kinase inhibitors (genistein and erbstatin analog) and an inhibitor (2'-amino-3'-methoxyflavone; PD98059) of the mitogen-activated protein (MAP) kinase kinase on the primary granule exocytosis and superoxide (O2.-) production of human neutrophil granulocytes were compared. The effector responses induced by stimulation of the chemotactic receptors by formyl-methionyl-leucyl-phenylalanine and platelet-activating factor were blocked both by genistein and erbstatin analog. In contrast, degranulation and O2.- production triggered by the activation of protein kinase C with phorbol-12-myristate-13-acetate were reduced by erbstatin analog but not by genistein. This inhibitory pattern was observed in both effector responses, but the sensitivity of O2.- production toward tyrosine kinase inhibition was markedly higher than that of degranulation. PD98059 caused no considerable effect on any of the above responses. The data presented indicate that tyrosine kinases are involved not only in the respiratory burst but also in the organization of the degranulation response of neutrophil granulocytes. It is suggested that several tyrosine kinases of different inhibitor sensitivity may participate in the transduction of extracellular signals. However, activation of the MAP kinase cascade does not appear to be involved in either of the investigated biological responses of the neutrophils.

Regulation of capacitative Ca2+ influx in human neutrophil granulocytes. Alterations in chronic granulomatous disease.

Ca2+ entry through the capacitative (store-regulated) pathway was shown to be inhibited in neutrophil granulocytes by the protein kinase C activator phorbol 12-myristate 13-acetate and the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) by a hitherto unknown mechanism. Measuring both Ca2+ and Mn2+ entry into store-depleted cells we show in the present study that inhibition of the capacitative pathway is absent in various forms of chronic granulomatous disease. To establish the possible relationship between inhibition of the capacitative pathway and ability of O-2 production and consequent membrane depolarization, gradual changes of the membrane potential were evoked in neutrophils of healthy individuals. This was accomplished by pharmacological manipulation of the membrane potential and by variations of the concentration and type of the stimulant. Close relationship was observed between membrane depolarization and inhibition of Mn2+ entry through the capacitative transport route. Our results provide an explanation for the inhibitory action of fMLP and phorbol 12-myristate 13-acetate on capacitative cation influx and reveal that upon physiological stimulation, Ca2+ entry into neutrophils is restricted by the depolarization accompanying O-2 production.

In vitro activation of the NADPH oxidase by fluoride. Possible involvement of a factor activating GTP hydrolysis on Rac (Rac-GAP).

The possible mechanism of activation of the NADPH oxidase by fluoride was investigated in the cell-free system. It is shown that the stimulatory effect of fluoride is inhibited by guanosine 5'-O-(2-thiodiphosphate) (GDP[S]) and potentiated by GTP. The effect of fluoride is not additive with GTP[S]. Fluoride activation requires the presence of Mg2+ in millimolar concentration but is independent of Al3+. The activating effect of fluoride is preserved in solubilized membrane extract after removal of the majority of heterotrimeric GTP-binding proteins by immunoadsorption. Fluoride has no direct action either on the nucleotide exchange of GTP hydrolysis of the isolated Rac protein. In contrast, fluoride effectively inhibits Rac-GTPase activity enhanced by a membrane component. In this way, fluoride could prolong the prevalence of Rac in the GTP-bound state and, as a consequence, activate NADPH oxidase. The possibility of the involvement of a membrane-bound Rac GTPase-activating protein activity in the physiological regulation of the enzyme is raised.

Evidence for ADP-ribosylation-factor-mediated activation of phospholipase D by m3 muscarinic acetylcholine receptor.

Activation of phospholipase D (PLD) is a cellular response to a wide variety of extracellular ligands. However, the exact mechanisms that link cell surface receptors to PLD remain unclear. In this study, we report the involvement of the small-molecular-mass guanine-nucleotide-binding protein, ADP-ribosylation factor (ARF), in the activation of PLD by the muscarinic acetylcholine receptor (mAChR) in human embryonic kidney cells stably expressing the human m3 subtype. PLD stimulation in permeabilized cells by guanosine 5'-O-[gamma-thio]triphosphate (GTP[S]) was dependent on a cytosolic factor and reconstituted by purified recombinant ARF 1. Brefeldin A, a known inhibitor of the ARF guanine-nucleotide-exchange-factor activity in Golgi membranes, inhibited mAChR-stimulated PLD, whereas basal PLD activity and stimulation by GTP[S] were not affected. Upon cell permeabilization without the addition of stimulus, ARF proteins were released. However, the addition of GTP[S] during permeabilization and mAChR activation before permeabilization caused an almost complete and partial (about 60%) inhibition, respectively, of ARF release, indicating that ARF proteins are activated and thereby translocated to membranes. The results indicate that ARF proteins and their nucleotide-exchange factor are apparently involved in the signalling pathway leading from mAChR activation to PLD stimulation in human embryonic kidney cells.

Thapsigargin inhibits Ca2+ entry into human neutrophil granulocytes.

The mechanism of Ca2+ entry after ligand binding to receptors on the surface of non-excitable cells is a current focus of interest. Considerable attention has been given to Ca2+ influx induced by emptying of intracellular pools. Thapsigargin, an inhibitor of microsomal Ca(2+)-ATPase, is an important tool in inducing store-regulated Ca2+ influx. In the present paper we show that, at concentrations above 500 nM, thapsigargin also has an opposite effect: it inhibits store-regulated Ca2+ influx into Fura-2-loaded human neutrophil granulocytes. As thapsigargin has been frequently applied at concentrations up to 2 microM, its inhibitory action on plasma-membrane Ca2+ fluxes deserves consideration.